Recent evidence has accumulated that coagulation factor Xl (FXl) has an essential role both In maintaining normal hemostasis and in the pathogenesis of thrombosis, and that inhibition of FXI function might prevent the formation of pathological thrombi. A large amount of information has accumulated in recent years concerning the biochemistry, molecular biology and physiology of the zymogen (FXI) and the genetics of FXI-deficiency. Thus a great deal of information is available concerning the synthesis of the protein, its site and mechanisms of activation, its cellular receptor, and the molecular domains within the amino-terminal tandem repeats (Apple domains) that mediate many of its protein-protein and protein-receptor interactions. In marked contrast, very little information is available about the biochemistry and physiology of the enzyme (FXla), the mechanism of FXla-catalyzed FIX-activation and the molecular structures within the carboxy-terminal catalytic domain that mediate substrate (FIX) recognition, cellular receptor (platelet) binding and regulation by physiological inhibitors such as protease nexin-2 (PN2) and heparin. The long-term goals of the present proposal are to elucidate the molecular mechanisms involved in the interaction of FXla with its substrate, its cellular (platelet) receptor(s) and its regulators and to define the structural biology mediating the exposure of exosites within the catalytic domain that result from the conversion of the zymogen to the protease. Specifically we propose: 1) To prepare constitutively inactive (zymogen-like), constitutively active (enzyme-like) and active-site-inhibited forms of the FXla catalytic domain and characterize them with respect to their functional interactions with the substrate, FIX; and to determine the structure and functional characteristics of the FXla catalytic domain extended macromolecular substrate recognition exosite(s) that mediate(s) the interaction of FXla with FIX. 2) To determine the structure and functional characteristics of the FXla catalytic domain exosite(s) that mediate(s) its interaction with the Kunitz protease inhibitor (KPI) domain of PN2. 3) To determine the structural and functional characteristics of the FXla catalytic domain exosite(s) that mediate(s) its interaction with heparin. 4) To determine the structural and functional characteristics of the FXla catalytic domain exosite(s) that mediate(s) its interaction with activated platelets.